Little-by-little, step-by-step, and drop-by-drop, we are beginning to appreciate the vital importance of intracellular liquid-like droplet formation to fundamental mechanisms such as epigenetic inheritance and heterochromatin domain formation. Liquid-like droplets aid these processes by biological “phase-separation” or the selective concentration of some molecules into a small intracellular area and the exclusion of others. Excitingly, two new papers from the labs of Richard A. Young (Whitehead Institute for Biomedical Research) and Ibrahim I. Cissé (Massachusetts Institute of Technology, Cambridge, MA, USA) now demonstrate that the formation of droplet-like “condensates” in embryonic stem cells (ESCs) provides a previously undescribed means of controlling gene expression and may play a vital role in cell fate determination.
Controlling Cell Identity via Coactivator Condensates at Super-Enhancer Elements
The first study from Sabari and colleagues assessed the characteristics of the high-density transcriptional machinery present at clusters of enhancers that regulate the expression of cell type-specific genes, known as super-enhancers (SE). As previous reports had observed sharp transitions between the formation and dissolution of the transcriptional machinery at SEs, the authors proposed a role for phase separation in this process in mouse ESCs (mESCs).
Here´s a blow-by-blow account of their exciting new study:
- Fixed cell immunofluorescence and live cell fluorescence microscopy established the formation of distinct puncta formed by SE-related coactivators (Bromodomain-containing protein 4 (BRD4) and Mediator of RNA polymerase II transcription subunit 1 (MED1)) within mESC nuclei
- Immunofluorescence analysis also indicated that the BRD4 and MED1 coactivator puncta associate with SEs for cell fate genes
- Fluorescence recovery after photobleaching (FRAP) assays demonstrated that BRD4 and MED1 coactivator puncta exhibit liquid-like rates of fluorescence recovery consistent with phase-separated liquid-like condensates
- Treatment with 1,6-hexanediol, a compound known to disrupt liquid-like condensates, disrupted BRD4 and MED1 coactivator puncta, confirming a liquid-like condensate nature
- Chromatin immunoprecipitation-sequencing (ChIP-seq) for RNA polymerase II (RNAPII) across SE-associated gene bodies linked condensate integrity loss following 1,6-hexanediol treatment to transcriptional inhibition
- Droplet formation assays established that the intrinsically disordered regions (IDRs) of the BRD4 and MED1 proteins help to form the phase-separated droplets in vitro
- The authors confirmed IDR-mediated droplet formation in vivo using a previously developed optoIDR assay that examines IDR-dependent, light-inducible droplet formation
- Furthermore, MED1-IDR droplets compartmentalized and concentrated the apparatus required for transcription from nuclear extracts, such as BRD4 and the largest subunit of RNAPII (RPB1)
Richard Young, the senior author of this study shares, “These findings challenge our conventional views of gene control, how the genome is organized, and the fundamental operating systems of cells.” He also adds “It’s interesting to consider whether the biophysical properties of condensates may be responsible for other observations in regulatory biology.”
Advanced Imaging Acclaims Condensate-Mediated Coordination of Cell Fate Gene Expression
Our second study from Cho and Colleagues sought to analyze the spatial organization and dynamics of transcriptional machinery by the interrogation of the Mediator complex and RNA Polymerase II (Pol II) holoenzyme in mESCs using state-of-the-art imaging techniques (live cell super-resolution imaging and light sheet imaging). Previous studies had suggested that Mediator interacts with Pol II at gene promoters; however, there was no prior evidence of this direct interaction in living cells. Excitingly, while exploring the Mediator-Pol II association, the team also uncovered more evidence that the formation of the liquid-like transcriptional condensates helps to coordinate cell fate gene expression.
Let us guide you step-by-step through these thrilling findings:
- Imaging analysis suggested that Mediator and Pol II formed both small transient clusters in mESCs and larger more stable clusters
- The stable clusters decreased in size and number during mESC differentiation, suggesting a link to cell fate determination
- Mediator and Pol II colocalized within the large stable clusters
- Treatment with JQ1, a drug that causes loss of BRD4 from enhancer chromatin, dissolved Mediator and Pol II clusters and suggested that Mediator and Pol II clusters form at SEs
- Imaging analysis indicated that stable clusters coalesce upon contact with biophysical dynamics reminiscent of biomolecular condensates
- FRAP and 1,6-hexanediol sensitivity analyses further demonstrated the liquid-like condensate nature of Mediator and Pol II clusters
- The authors confirmed a model by which Mediator and Pol II transcriptional condensates form around chromatin (via tracking diffusion dynamics) and colocalize with enhancer-controlled active genes such as Esrrb (via RNA labeling assays and lattice light sheet microscopy)
Overall, this second study confirms the previous findings, thereby firmly establishing liquid-like condensates as fundamental coordinators of cell-fate associated gene expression.
Epigenetic Inheritance, Heterochromatin Formation, and Cell Fate; What’s Next?
Taken together, these two studies confirm the critical nature of phase separation and liquid-like droplet formation to essential biological processes. So what’s next!? To find out, keep your eyes on Epigenie.com in the coming months and years!
But if you are hungry for more, read all about how BRD4 and MED1 condensates and the transcriptional control of cell fate genes in mESCs at Science, June 2018, and discover all the details on how Mediator and Pol II condensates control gene expression and cell fate at Science, June 2018